NREL is a national laboratory of the U.S. Department of Energy, Office of Energy 

Efficiency & Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.

Contract No. DE-AC36-08GO28308

Determination of Structural 

Carbohydrates and Lignin in 

Biomass

Laboratory Analytical Procedure (LAP)

Issue Date: April 2008 

Revision Date: August 2012

(Version 08-03-2012)

A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, 

J. Sluiter, D. Templeton, and D. Crocker 

Technical Report

NREL/TP-510-42618

Revised August 2012
NREL is a national laboratory of the U.S. Department of Energy, Office of Energy 

Efficiency & Renewable Energy, operated by the Alliance for Sustainable Energy, LLC.

National Renewable Energy Laboratory

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Golden, Colorado 80401

303-275-3000 • www.nrel.gov

Contract No. DE-AC36-08GO28308

Determination of Structural 

Carbohydrates and Lignin in 

Biomass

Laboratory Analytical Procedure 

(LAP) 

Issue Date: April 2008 

Revision Date: August 2012 

(Version 08-03-2012) 

A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, 

J. Sluiter, D. Templeton, and D. Crocker 

NREL Laboratory Analytical Procedures for standard biomass 

analysis are available electronically at 

http://www.nrel.gov/biomass/analytical_procedures.html

Technical Report

NREL/TP-510-42618

Revised August 2012
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Printed on paper containing at least 50% wastepaper, including 10% post consumer waste. 
1 

Procedure Title: Determination of Structural Carbohydrates and Lignin in 

Biomass

Laboratory Analytical Procedure

1. Introduction

1.1 Carbohydrates and lignin make up a major portion of biomass samples. These constituents 

must be measured as part of a comprehensive biomass analysis. Carbohydrates can be 

structural or non-structural. Structural carbohydrates are bound in the matrix of the biomass, 

while non-structural carbohydrates can be removed using extraction or washing steps. Lignin 

is a complex phenolic polymer.

1.2 Portions of this procedure are substantially similar to ASTM E1758-01 “Standard Method for 

the Determination of Carbohydrates by HPLC.”

1.3 This procedure is suitable for samples that do not contain extractives. This procedure uses a 

two-step acid hydrolysis to fractionate the biomass into forms that are more easily quantified.

The lignin fractionates into acid insoluble material and acid soluble material. The acid 

insoluble material may also include ash and protein, which must be accounted for during 

gravimetric analysis. The acid soluble lignin is measured by UV-Vis spectroscopy. During 

hydrolysis the polymeric carbohydrates are hydrolyzed into the monomeric forms, which are 

soluble in the hydrolysis liquid. They are then measured by HPLC. Protein may also partition 

into the liquid fraction. A measure of acetyl content is necessary for biomass containing 

hemicellulose with a xylan backbone, but not biomass containing a mannan backbone. Acetate 

is measured by HPLC.

2. Scope

2.1 This procedure is appropriate for extractives free biomass, which includes biomass that has 

been extracted using LAP “Determination of Extractives in Biomass,” as well as process solids 

containing no extractives. Results are reported on an oven dry weight basis. Results may be 

reported on an as received biomass basis or an extractives free basis, depending on type of 

biomass used. LAP “Preparation of Samples for Biomass Compositional Analysis” should be 

used prior to this procedure. 

2.2 This procedure is appropriate for biomass containing the components listed throughout the 

procedure. Any biomass containing other interfering components not listed must be further 

investigated.

2.3 A measure of acetyl content is necessary for biomass containing hemicellulose with a xylan 

backbone, but not biomass containing a mannan backbone. 

2.4 All analyses should be performed in accordance with an appropriate laboratory specific Quality 

Assurance Plan (QAP).

3. Terminology 

3.1 Oven dry weight (ODW)—the weight of biomass mathematically corrected for the amount of 
2 

moisture present in the sample at the time of weighing.

3.2 Prepared biomass—biomass prepared according to LAP “Preparation of Samples for Biomass 

Compositional Analysis.” 

3.3 Extractives free biomass—biomass after exhaustive water and ethanol extraction (refer to LAP 

“Determination of Extractives in Biomass”).

3.4 Acid insoluble lignin—the residue remaining on a medium porosity filtering crucible after a two 

step hydrolysis, with correction for acid insoluble ash and acid insoluble protein, if necessary.

3.5 Structural carbohydrates—polymeric carbohydrates, namely cellulose and hemicellulose.

3.6 Non-structural components—non-chemically bound components of biomass that include but 

are not limited to sucrose, nitrate/nitrites, protein, ash, chlorophyll, and waxes.

4. Significance and Use

4.1 This procedure is used, possibly in conjunction with other procedures, to determine the amount 

of structural carbohydrates and lignin in a solid biomass sample. 

5. Interferences

5.1 This procedure has been optimized for the particle size range specified in LAP “Preparation of 

Samples for Biomass Compositional Analysis.” Deviation to a smaller particle size may result 

in a low bias in carbohydrate content (and consequent high lignin bias) due to excessive 

carbohydrate degradation. Deviation to a larger particle size may also result in a low bias in 

carbohydrate content (and consequent high lignin bias) due to incomplete hydrolysis of 

polymeric sugars to monomeric sugars.

5.2 Samples containing extractives are not suitable for this procedure. Extractives will partition 

irreproducibly, resulting in a high lignin bias. 

5.3 Samples with an ash content above 10 wt % may not be suitable for this procedure, as the 

sample may contain soil or other minerals that will interfere with appropriate acid 

concentrations and may catalyze side reactions.

5.4 Samples with a moisture content above 10 wt % may not be suitable for this procedure, as the 

excess moisture will interfere with appropriate acid concentrations. Samples should be dried 

(air-dried or oven dried at less than 40°C) prior to this procedure. 

5.5 Samples containing protein will bias the acid insoluble lignin high unless the protein is 

accounted for in the gravimetric determination of acid insoluble material. An independent 

nitrogen analysis is required to estimate the protein content of the residue. The protein estimate 

is then subtracted from the acid insoluble residue measurement. Physical separation of the acid 

insoluble protein from the acid insoluble lignin is beyond the scope of this procedure.

5.6 This procedure is not suitable for samples containing added acid, base, or catalyst. 

5.7 Certain guard columns for carbohydrate quantification may cause artifact peaks. Individual 

carbohydrates should be run on new columns and guard columns to verify to absence of artifact 

peaks.

6. Apparatus

6.1 Analytical balance, accurate to 0.1 mg.

6.2 Convection drying oven, with temperature control of 105 ± 3°C. 

6.3 Muffle furnace, equipped with a thermostat, set to 575 ± 25 °C or equipped with optional 

ramping program. 

6.4 Water bath, set at 30 ± 3 °C. 

6.5 Autoclave, suitable for autoclaving liquids, set to 121 ± 3 °C. 

6.6 Filtration setup, equipped with a vacuum source and vacuum adaptors for crucibles.
3 

6.7 Desiccator containing desiccant.

6.8 HPLC system equipped with refractive index detector and the following columns:

6.8.1 Shodex sugar SP0810 or Biorad Aminex HPX-87P column (or equivalent) with ionic 

form H+/CO3

-

 deashing guard column. 

6.8.2 Biorad Aminex HPX-87H column (or equivalent) equipped with an appropriate guard 

column.

6.9 UV-Visible spectrophotometer, diode array or single wavelength, with high purity quartz 

cuvettes of pathlength 1 cm. 

6.10Automatic burette, capable of dispensing 84.00 mL water, optional.

7. Reagents and Materials

7.1 Reagents

7.1.1 Sulfuric acid, 72% w/w (specific gravity 1.6338 at 20°C) – (also commercially available 

as a reagent for the determination of fluorine, from Fluka #00647). 

7.1.2 Calcium carbonate, ACS reagent grade. 

7.1.3 Water, purified, 0.2 μm filtered. 

7.1.4 High purity standards: D-cellobiose, D(+)glucose, D(+)xylose, D(+)galactose, 

L(+)arabinose, and D(+)mannose. 

7.1.5 Second set of high purity standards, as listed above, from a different source 

(manufacturer or lot), to be used to prepare calibration verification standards (CVS).

7.2 Materials

7.2.1 QA standard, well characterized, such as a National Institute of Standards and 

Technology (NIST) biomass standard or another well characterized sample of similar 

composition to the samples being analyzed. 

7.2.2 Pressure tubes, minimum 90 mL capacity, glass, with screw on Teflon caps and o-ring 

seals (Ace glass # 8648-30 tube with #5845-47 plug, or equivalent). 

7.2.3 Teflon stir rods sized to fit in pressure tubes and approximately 5 cm longer than 

pressure tubes. 

7.2.4 Filtering crucibles, 25 mL, porcelain, medium porosity, Coors #60531 or equivalent. 

7.2.5 Bottles, wide mouth, 50 mL. 

7.2.6 Filtration flasks, 250 mL. 

7.2.7 Erlenmeyer flasks, 50 mL. 

7.2.8 Adjustable pipettors, covering ranges of 0.02 to 5.00 mL and 84.00 mL. 

7.2.9 pH paper, range 4–9. 

7.2.10 Disposable syringes, 3 mL, fitted with 0.2 μm syringe filters. 

7.2.11 Autosampler vials with crimp top seals to fit.

8. ES&H Considerations and Hazards

8.1 Sulfuric acid is corrosive and should be handled with care.

8.2 Use caution when handling hot pressure tubes after removal from the autoclave, as the 

pressurized tubes can cause an explosion hazard. 

8.3 When placing crucibles in a furnace or removing them, use appropriate personal protective 

equipment, including heat resistant gloves. 

8.4 Operate all equipment in accordance with the manual and NREL Safe Operating Procedures. 

8.5 Follow all applicable NREL chemical handling procedures.

9. Sampling, Test Specimens, and Test Units

9.1 Care must be taken to ensure a representative sample is taken for analysis.

9.2 LAP “Preparation of Samples for Biomass Compositional Analysis” should be performed 
4 

prior to this analysis. Samples must have a minimum total solids content of 85%.

9.3 LAP “Determination of Extractives in Biomass” should be performed prior to this analysis if 

extractives are present in the sample.

9.4 LAP “Determination of Solids in Biomass” should be performed at the same time that 

samples for this analysis are weighed out. 

9.5 This procedure is suitable for samples that have been air dried or lyophilized. Samples dried at 

a temperature of 45°C or higher are not suitable for this procedure. 

9.6 Steps 9.2 to 9.4 should be applied to the QA standard.

10. Procedure

10.1 Prepare the sample for analysis and hydrolyze.

10.1.1 Place an appropriate number of filtering crucibles in the muffle furnace at 575 ± 25 °C 

for a minimum of four hours. Remove the crucibles from the furnace directly into a 

desiccator and cool for a specific period of time, one hour is recommended. Weigh the 

crucibles to the nearest 0.1 mg and record this weight. It is important to keep the 

crucibles in a specified order, if they are not marked with identifiers. Permanent marking 

decals are available from Wale Apparatus. Do not mark the bottom of the filtering 

crucible with a porcelain marker, as this will impede filtration.

10.1.2 Place the crucible back into the muffle furnace at 575 ± 25 °C and ash to constant 

weight. Constant weight is defined as less than ± 0.3 mg change in the weight upon one 

hour of re-heating the crucible.

10.1.3 Weigh 300.0 ± 10.0 mg of the sample or QA standard into a tared pressure tube. Record 

the weight to the nearest 0.1 mg. Label the pressure tube with a permanent marker. LAP 

“Determination of Total Solids in Biomass” should be performed at the same time, to 

accurately measure the percent solids for correction. Each sample should be analyzed in 

duplicate, at minimum. The recommended batch size is three to six samples and a QA 

standard, all run in duplicate. 

10.1.4 Add 3.00 ± 0.01 mL (or 4.92 ± 0.01 g) of 72% sulfuric acid to each pressure tube. Use a 

Teflon stir rod to mix for one minute, or until the sample is thoroughly mixed. 

10.1.5 Place the pressure tube in a water bath set at 30 ± 3 °C and incubate the sample for 60 ± 

5 minutes. Using the stir rod, stir the sample every 5 to 10 minutes without removing the 

sample from the bath. Stirring is essential to ensure even acid to particle contact and 

uniform hydrolysis. 

10.1.6 Upon completion of the 60-minute hydrolysis, remove the tubes from the water bath. 

Dilute the acid to a 4% concentration by adding 84.00 ± 0.04 mL deionized water using 

an automatic burette. Dilution can also be done by adding 84.00 ± 0.04 g of purified 

water using a balance accurate to 0.01 g. Screw the Teflon caps on securely. Mix the 

sample by inverting the tube several times to eliminate phase separation between high 

and low concentration acid layers. 

Note: The volume of the 4% solution will be 86.73 mL, as demonstrated in the 

following calculations.

Density 72% H2SO4= d72% H2SO4= 1.6338 g/mL

Density H2O= dH2O= 1.00 g/mL

Density 4% H2SO4 = d4% H2SO4 = 1.025 g/mL

1. The weight of 3.00 mL 72% H2SO4 is:

3.00 mL 72% H2SO4 x d72% H2SO4= 4.90 g 72% H2SO4 

2. The composition of 3 mL of 72% H2SO4 is: 
5 

4.90 g 72% H2SO4 x 72% (acid wt) = 3.53 g acid

4.90 g 72% H2SO4 x 28% (water wt) = 1.37 g water 

3. The concentration of H2SO4 after dilution is:

3.53 g acid / (84.00 g H2O + 4.90 g 72% H2SO4) = 3.97 % H2SO4 (w/w)

4. The total volume of solution present after dilution is: 

(4.90 g H2SO4 + 84.00 g H2O) x (d4% H2SO4)

-1 = 86.73 mL

10.1.7 Prepare a set of sugar recovery standards (SRS) that will be taken through the remaining 

hydrolysis and used to correct for losses due to destruction of sugars during dilute acid 

hydrolysis. SRS should include D-(+)glucose, D-(+)xylose, D-(+)galactose, 

-L(+)arabinose, and D-(+)mannose. SRS sugar concentrations should be chosen to most 

closely resemble the concentrations of sugars in the test sample. Weigh out the required 

amounts of each sugar, to the nearest 0.1 mg, and add 10.0 mL deionized water. Add 348 

μL of 72% sulfuric acid. Transfer the SRS to a pressure tube and cap tightly.

10.1.7.1 A fresh SRS is not required for every analysis. A large batch of sugar recovery 

standards may be produced, filtered through 0.2 μm filters, dispensed in 10.0 mL 

aliquots into sealed containers, and labeled. They may be stored in a freezer and 

removed when needed. Thaw and vortex the frozen SRS prior to use. If frozen SRS 

are used, the appropriate amount of acid must be added to the thawed sample and 

vortexed prior to transferring to a pressure tube.

10.1.8 Place the tubes in an autoclave safe rack, and place the rack in the autoclave. Autoclave 

the sealed samples and sugar recovery standards for one hour at 121°C, usually the 

liquids setting. After completion of the autoclave cycle, allow the hydrolyzates to slowly 

cool to near room temperature before removing the caps. (If step 10.2 is not performed, 

draw a 10 mL aliquot of the liquor for use in step 10.5.)

10.2 Analyze the sample for acid insoluble lignin as follows.

10.2.1 Vacuum filter the autoclaved hydrolysis solution through one of the previously weighed 

filtering crucibles. Capture the filtrate in a filtering flask.

10.2.2 Transfer an aliquot, approximately 50 mL, into a sample storage bottle. This sample will 

be used to determine acid soluble lignin as well as carbohydrates, and acetyl if 

necessary. Acid soluble lignin determination must be done within 6 hours of hydrolysis. 

If the hydrolysis liquor must be stored, it should be stored in a refrigerator for a 

maximum of two weeks. It is important to collect the liquor aliquot before proceeding to 

step 10.2.3. 

10.2.3 Use deionized water to quantitatively transfer all remaining solids out of the pressure 

tube into the filtering crucible. Rinse the solids with a minimum of 50 mL fresh 

deionized water. Hot deionized water may be used in place of room temperature water to 

decrease the filtration time.

10.2.4 Dry the crucible and acid insoluble residue at 105 ± 3 °C until a constant weight is 

achieved, usually a minimum of four hours. 

10.2.5 Remove the samples from the oven and cool in a desiccator. Record the weight of the 

crucible and dry residue to the nearest 0.1 mg. 

10.2.6 Place the crucibles and residue in the muffle furnace at 575 ± 25 °C for 24 ± 6 hours.

10.2.6.1 A furnace with temperature ramping may also be used.

Furnace Temperature Ramp Program:

Ramp from room temperature to 105 °C 

Hold at 105 °C for 12 minutes
6 

Ramp to 250 °C at 10 °C / minute

Hold at 250 °C for 30 minutes 

Ramp to 575 °C at 20 °C / minute

Hold at 575 °C for 180 minutes 

Allow temperature to drop to 105 °C 

Hold at 105 °C until samples are removed

10.2.7 Carefully remove the crucible from the furnace directly into a desiccator and cool for a 

specific amount of time, equal to the initial cool time of the crucibles. Weigh the 

crucibles and ash to the nearest 0.1 mg and record the weight. Place the crucibles back in 

the furnace and ash to a constant weight. (The amount of acid insoluble ash is not equal 

to the total amount of ash in the biomass sample. Refer to LAP “Determination of Ash in 

Biomass” if total ash is to be determined.)

10.3 Analyze the sample for acid soluble lignin as follows.

10.3.1 On a UV-Visible spectrophotometer, run a background of deionized water or 4% sulfuric 

acid. 

10.3.2 Using the hydrolysis liquor aliquot obtained in step 10.2.2, measure the absorbance of 

the sample at an appropriate wavelength on a UV-Visible spectrophotometer. Refer to 

section 11.3 for suggested wavelength values. Dilute the sample as necessary to bring the 

absorbance into the range of 0.7–1.0, recording the dilution. Deionized water or 4% 

sulfuric acid may be used to dilute the sample, but the same solvent should be used as a 

blank. Record the absorbance to three decimal places. Reproducibility should be ± 0.05 

absorbance units. Analyze each sample in duplicate, at minimum. (This step must be 

done within six hours of hydrolysis.) 

10.3.3 Calculate the amount of acid soluble lignin present using calculation 11.3. 

10.4 Analyze the sample for structural carbohydrates.

10.4.1 Prepare a series of calibration standards containing the compounds that are to be 

quantified, referring to Table 1 for suggested concentration range. Use a four point 

calibration. If standards are prepared outside of the suggested ranges, the new range for 

these calibration curves must be validated.

10.4.1.1 Table 1. Suggested concentration ranges for 10.4.1 calibration standards

Component Suggested 

concentration range 

(mg/ml)

D-cellobiose 0.1–4.0

D(+)glucose 0.1–4.0

D(+)xylose 0.1–4.0

D(+)galactose 0.1–4.0

L(+)arabinose 0.1–4.0

D(+)mannose 0.1–4.0

CVS Middle of linear range, 

concentration not equal 

to a calibration point 

(2.5 suggested)

10.4.1.2 A fresh set of standards is not required for every analysis. A large batch of 

standards may be produced, filtered through 0.2 μm filters into autosampler 
7 

vials, sealed and labeled. The standards and CVS samples may be stored in a 

freezer and removed when needed. Thaw and vortex frozen standards prior to 

use. During every use, standards and CVS samples should be observed for 

unusual concentration behavior. Unusual concentrations may mean that the 

samples are compromised or volatile components have been lost. Assuming 

sufficient volume, standards and CVS samples should not have more than 12 

injections drawn from a single vial. In a chilled autosampler chamber, the 

lifetime of standards and CVS samples is approximately three to four days.

10.4.2 Prepare an independent calibration verification standard (CVS) for each set of calibration 

standards. Use reagents from a source or lot other than that used in preparing the 

calibration standards. Prepare the CVS at a concentration that falls in the middle of the 

validated range of the calibration curve. The CVS should be analyzed on the HPLC after 

each calibration set and at regular intervals throughout the sequence, bracketing groups 

of samples. The CVS is used to verify the quality and stability of the calibration curve(s) 

throughout the run. 

10.4.3 Using the hydrolysis liquor obtained in step 10.2.2, transfer an approximately 20 mL 

aliquot of each liquor to a 50 mL Erlenmeyer flask. 

10.4.4 Use calcium carbonate to neutralize each sample to pH 5–6. Avoid neutralizing to a pH 

greater than 6 by monitoring with pH paper. Add the calcium carbonate slowly after 

reaching a pH of 4. Swirl the sample frequently. After reaching pH 5–6, stop calcium 

carbonate addition, allow the sample to settle, and decant off the supernatant. The pH of 

the liquid after settling will be approximately 7. (Samples should never be allowed to 

exceed a pH of 9, as this will result in a loss of sugars.) 

10.4.5 Prepare the sample for HPLC analysis by passing the decanted liquid through a 0.2 μm 

filter into an autosampler vial. Seal and label the vial. Prepare each sample in duplicate, 

reserving one of the duplicates for analysis later if necessary. If necessary, neutralized 

samples may be stored in the refrigerator for three or four days. After this time, the 

samples should be considered compromised due to potential microbial growth. After 

cold storage, check the samples for the presence of a precipitate. Samples containing a 

precipitate should be refiltered, while still cold, through a 0.2 μm filter.

10.4.6 Analyze the calibration standards, CVS, and samples by HPLC using a Shodex sugar 

SP0810 or Biorad Aminex HPX-87P column equipped with the appropriate guard 

column. 

HPLC conditions: 

Injection volume: 10–50 μL, dependent on concentration and detector limits 

Mobile phase: HPLC grade water, 0.2 μm filtered and degassed

Flow rate: 0.6 mL/minute

Column temperature: 80–85 °C 

Detector temperature: as close to column temperature as possible

Detector: refractive index

Run time: 35 minutes

Note: The deashing guard column should be placed outside of the heating unit and 

kept at ambient temperature. This will prevent artifact peaks in the chromatogram.

10.4.7 Check test sample chromatograms for presence of cellobiose and oligomeric sugars.

Levels of cellobiose greater than 3 mg/mL indicate incomplete hydrolysis. Fresh samples 

should be hydrolyzed and analyzed.
8 

10.4.8 Check test sample chromatograms for the presence of peaks eluting before cellobiose 

(retention time of 4–5 minutes using recommended conditions). These peaks may 

indicate high levels of sugar degradation products in the previous sample, which is 

indicative of over hydrolysis. All samples from batches showing evidence of overhydrolysis should have fresh samples hydrolyzed and analyzed.

10.5 Analyze the sample for acetyl content if necessary.

10.5.1 Prepare 0.005 M (0.01 N) sulfuric acid for use as a HPLC mobile phase. In a 2 L 

volumetric flask, add 2.00 mL of standardized 10 N sulfuric acid and bring to volume 

with HPLC grade water. Filter through a 0.2 μm filter and degas before use. If 10N 

sulfuric acid is not available, concentrated sulfuric acid may also be used. 278 μL 

concentrated sulfuric acid brought to volume in a 1 L volumetric flask with HPLC grade 

water will also produce 0.005 M sulfuric acid. 

10.5.2 Prepare a series of calibration standards containing the compounds that are to be 

quantified. Acetic acid is recommended, formic acid and levulinic acid are optional. A 

range of 0.02 to 0.5 mg/mL is suggested. An evenly spaced four point calibration is 

suggested. If standards are prepared outside of the suggested ranges, the new range for 

these calibration curves must be validated.

10.5.3 Prepare an independent calibration verification standard (CVS) for each set of calibration 

standards, using components obtained from a source other than that used in preparing the 

calibration standards. The CVS must contain precisely known amounts of each 

compound contained in the calibration standards, at a concentration that falls in the 

middle of the validated range of the calibration curve. The CVS should be analyzed on 

the HPLC after each calibration set and at regular intervals throughout the sequence, 

bracketing groups of samples. The CVS is used to verify the quality and stability of the 

calibration curve(s) throughout the run. 

10.5.4 Prepare the sample for HPLC analysis by passing a small aliquot of the liquor collected 

in step 10.2.2 through a 0.2 μm filter into an autosampler vial. Seal and label the vial. If 

it is suspected that the sample concentrations may exceed the calibration range, dilute the 

samples as needed, recording the dilution. The concentrations should be corrected for 

dilution after running. 

10.5.5 Analyze the calibration standards, CVS, and samples by HPLC using a Biorad Aminex 

HPX-87H column equipped with the appropriate guard column. 

HPLC conditions: 

Sample volume: 50 μL 

Mobile phase: 0.005 M sulfuric acid, 0.2 μm filtered and degassed

Flow rate: 0.6 mL/minute 

Column temperature: 55–65 °C 

Detector temperature: as close to column temperature as possible

Detector: refractive index

Run time: 50 minutes
9 

11. Calculations

11.1 Calculate the oven dry weight (ODW) of the extractives free sample, using the average total 

solids content as determined by the LAP “Standard Method for the Determination of Total 

Solids in Biomass.” 

100

 solidsTotal % x Weight ODW = sampledryair

11.2 Calculate and record the weight percent acid insoluble residue (AIR) and acid insoluble lignin 

(AIL) on an extractives free basis. 

100 x 

ODW

Weight Weight - = AIR %

sample

crucible plus AIR crucible

( ) ( ) 100 x 

ODW

Weight Weight - Weight Weight Weight - = AIL %

sample

crucible plus AIR crucible − crucible plus ash crucible − protein

Where: 

Weightprotein = Amount of protein present in the acid insoluble residue, as 

determined in LAP “Determination of Protein Content in Biomass.” This 

measurement is only necessary for biomass containing high amounts of protein. 

Our studies have shown that, for a corn stover feedstock, only a minor fraction of 

the protein condenses into the residue. For a pretreated corn stover, a significant 

amount of the nitrogenous material condenses into the AIR. The amount of nitrogen 

in the residue can be determined. If a nitrogen value is determined, the user should 

be aware that the nitrogen-to-protein factor will no longer be valid, as the 

nitrogenous material will likely have degraded. 

11.3 Calculate the amount of acid soluble lignin (ASL) on an extractives free basis. 

100 x 

xODW Pathlength

x Volume x UVabs Dilution

 = ASL %

sample

filtrate

ε ×

Where: 

UVabs = average UV-Vis absorbance for the sample at appropriate wavelength (see 

table below)

Volumehydrolysis liquor = volume of filtrate, 86.73 mL
10 

Dilution = 

Volumesample + Volume diluting solvent

Volumesample

ε = Absorptivity of biomass at specific wavelength (see table below)

ODWsample = weight of sample in milligrams

Pathlength = pathlength of UV-Vis cell in cm

Absorptivity constants for acid soluble lignin measurement for select biomass types

Biomass type Lambda max 

(nm)

Absorptivity at 

lambda max 

(L/g cm)

Recommended 

wavelength 

(nm)

Absorptivity at 

recommended 

wavelength 

(L/g cm)

Pinus RadiataNIST SRM 8493198 25 240 12

BagasseNIST SRM 8491198 40 240 25

Corn Stover- NREL 

supplied feedstock

198 55 320 30

Populus deltiodesNIST SRM 8492197 60 240 25

Note: Lambda max values often contain interfering peaks from carbohydrate degradation 

products. Recommended wavelength values have been chosen to minimize these 

interferences.

11.4 Calculate the total amount of lignin on an extractives free basis.

 % Lignin ASL %AIL % = ext free +

11.5 Calculate the total lignin value to an as received basis, if necessary

( ) ( )

100

 %100 Extractives % Ligninas received = Ligninext free

− % ×

Where: 

% Extractives = percent extractives in the prepared biomass sample, as determined 

in LAP “Determination of Extractives in Biomass”

11.6 Create a calibration curve for each analyte to be quantified using linear regression. From these 

curves, determine the concentration in mg/mL of each component present in the samples 

analyzed by HPLC, correcting for dilution if required. 

11.7 Calculate and record the amount of each calibration verification standard (CVS) recovered 

following HPLC analysis. 
11 

100 x 

 known conc.of standard,mg/mL

conc.detected by HPLC,mg/mL CVS % recovery =

11.8 For the sugar recovery standards (SRS), calculate the amount of each component sugar 

recovered after dilute acid hydrolysis, accounting for any dilution made prior to HPLC 

analysis. Average any replicate (%Rsugar) values obtained for each individual sugar and report 

%Ravg, sugar. 

100 x 

 known conc. sugarof before hydrolysis ,mg/mL

conc.detected by HPLC,mg/mL % R = sugar

11.9 Use the percent hydrolyzed sugar recovery values calculated in step 11.8 to correct the 

corresponding sugar concentration values obtained by HPLC for each of the hydrolyzed 

samples (Ccor. sample), accounting for any dilution made prior to HPLC analysis. 

 % R 100 / 

C x dilution factor C = ave. sugar

HPLC

x

Where: CHPLC = conc. of a sugar as determined by HPLC, mg/mL

% Rave. sugar = average recovery of a specific SRS component

Cx = Ccor. sample, concentration in mg/mL of a sugar in the hydrolyzed 

sample after correction for loss on 4% hydrolysis 

11.10Calculate the concentration of the polymeric sugars from the concentration of the 

corresponding monomeric sugars, using an anhydro correction of 0.88 (or 132/150) for C-5 

sugars (xylose and arabinose) and a correction of 0.90 (or162/180) for C-6 sugars (glucose, 

galactose, and mannose). 

anhydro C = C corr × Anhydro correction

Note: To be completely correct, the hydrolysis of the hemicellulose branches should be accounted 

for in the xylan values, since the loss of functional groups adds either a proton or a hydroxide to the 

xylan. Two examples of branch compounds that are quantified in this LAP are acetate and minor 

sugars such as galactan, arabinan, and mannan. However, for the typical concentrations of acetate 

and minor sugars seen in biomass samples, the resulting changes in xylan values are negligible, and 

so this correction is not performed. If desired, the correction can be applied per the equations 

below. However, this correction actually increases the uncertainty of the xylan value, since the 

uncertainties of the measured concentration values of the minor sugars are much larger than for 

xylose. It also makes the assumption that each of the minor sugars and acetate groups branch 

directly from the xylan backbone, which is likely an erroneous assumption. 

= ( (1−× − ×132/150)) Canhydro_xylan Xylosecorr XyloseAAcorrection XyloseMinorsugarcorrection
12 

Where:

(132 )

)(17

_

__

xyloseMW

OH MW

Xylose concentration

Acetic acid concentration XyloseAAcorrection

−

= ×

(132 )

)(1

_

__

xyloseMW

MWH

Xylose concentration

Minor sugar concentration XyloseMinorsugarcorrection

+

= ×

11.11Calculate the percentage of each sugar on an extractives free basis. 

100 x 

ODW

1000mg

1g V C

 = Sugar%

sample

anhydro filtrate

ext free

××

Where: Vfiltrate = volume of filtrate, 86.73 mL

11.12Calculate the percentage of each sugar on an as received basis, if necessary. 

( ) ( )

100

 %100 Extractives Sugar% Sugar = as received ext free

− % ×

Where: 

% Extractives = percent extractives in the prepared biomass sample, as determined 

in LAP “Determination of Extractives in Biomass”

11.13Calculate the acetate percentage on an extractives free basis.

% Acetateext free = 

CAA,HPLC x Volume filtrate x Conversion factor

ODWsample

 x 100

Where: 

CAA,HPLC = concentration in mg/mL of acetic acid as determined by HPLC

Volumehydrolysis liquor = volume of filtrate, generally 86.73 mL

Conversion factor = (59/60) = 0.983, the conversion from acetic acid to acetate in 

biomass 

Note (July 8, 2011): The conversion factor has been updated from previous versions 

of the LAP. The previous value was (43/60) = 0.717. 

11.14Calculate the acetyl percentage on an as received basis, if necessary.

( ) ( )

100

 %100 Extractives % Acetyl = Acetyl asreceived ext free

− % ×
13 

11.15To report or calculate the relative percent difference (RPD) between two samples, use the 

following calculation. 

RPD = 1 (X − X2)

X mean

 

   

  × 100

Where:

X1 and X2 = measured values

Xmean = the mean of X1 and X2 

11.16To report or calculate the root mean square deviation (RMS deviation) or the standard 

deviation (st dev) of the samples, use the following calculations. 

First find the root mean square (RMS) of the sample using: 

RMS = x m = mean = 

x

1

n

∑

 n 

 

 

 

 

 

  

 

 

 

 

 

  

2

Then find the root mean square deviation, or standard deviation, using: 

RMSdeviation = σ = stdev =

xi − x ( ) m

2

1

n

∑

n

Where:

xm = the root mean square of all x values in the set

n = number of samples in set 

xi = a measured value from the set

12. Report Format

12.1 Report the weight percent of lignin, each sugar, and acetate. Report on an as received basis, 

correcting for extractives if necessary.

12.2 For replicate analyses, report the average and relative percent difference.

13. Precision and Bias

13.1 Round robin testing – For a report documenting an international round robin test of biomass 

analysis methods, including this procedure, see Milne et al., 1992.

14. Quality Control

14.1 Reported significant figures or decimal places: Determined by data quality objectives and 

laboratory specific Quality Assurance Plan.
14 

14.2 Replicates: Run all samples in duplicate.

14.3 Blank: None. 

14.4 Relative percent difference criteria: Determined by data quality objectives and laboratory 

specific Quality Assurance Plan.

14.5 Calibration verification standard: Calibration verification standards should be independently 

prepared and analyzed as appropriate, per the procedure. 

14.6 Sample size: 4g, minimum, of sample, extracted if necessary (including amount necessary for 

percent solids determination). 

14.7 Sample storage: Hydrolysis liquors may be stored in a refrigerator for up to two weeks. 

Neutralized hydrolysis liquors may be stored in a refrigerator for up to four days. 

14.8 Standard storage: HPLC standards should be stored in a freezer and removed when needed. 

Thaw and vortex samples prior to use. 

14.9 Standard preparation: Standards should be prepared as described in the procedure, including a 

QA standard. 

14.10Definition of a batch: Any number of samples that are analyzed and recorded together. The 

recommended size is three to six samples with a QA standard, all run in duplicate.

14.11Control charts: All CVS, SRS recoveries, and NIST or QA standards should be control 

charted.

15. Appendices

15.1 None

16. References

16.1 NREL BAT Team Laboratory Analytical Procedure “Determination of Total Solids in 

Biomass.” 

16.2 NREL BAT Team Laboratory Analytical Procedure “Determination of Ash in Biomass.” 

16.3 NREL BAT Team Laboratory Analytical Procedure “Determination of Extractives in 

Biomass.” 

16.4 NREL CAT Task Laboratory Analytical Procedure#002 “Determination of Structural 

Carbohydrate Content in Biomass by High Performance Liquid Chromatography.” 

16.5 NREL CAT Task Laboratory Analytical Procedure#003 “Determination of Acid Insoluble 

Lignin in Biomass.” 

16.6 NREL CAT Task Laboratory Analytical Procedure#017 “Determination of o-Acetyl Groups 

in Biomass by High Performance Liquid Chromatography.” 

16.7 NREL CAT Task Laboratory Analytical Procedure#019 “Hydrolysis of Corn Stover for 

Compositional Analysis.”

16.8 Moore, W., and D. Johnson. 1967. Procedures for the Chemical Analysis of Wood and Wood 

Products. Madison, WI: U.S. Forest Products Laboratory, U.S. Department of Agriculture. 

16.9 ASTM E1758-01 “Standard method for the Determination of Carbohydrates by HPLC.” In 

2003 Annual Book of ASTM Standards, Volume 11.05. Philadelphia, PA: American Society 

for Testing and Materials, International 

16.10Milne, T. A.; Chum, H. L.; Agblevor, F. A.; Johnson, D. K. (1992). “Standardized Analytical 

Methods.” Biomass & Bioenergy. Proceedings of International Energy Agency Bioenergy 

Agreement Seminar, 2-3 April 1992, Edinburgh, U.K.. Vol. 2(1-6), 1992; pp. 341-366
15 

17. List of Revisions 

Version 08-03-2012: 

• Page 11: Section 11.10 includes notes on the correction of the xylan number for minor 

sugars and acetate, as well as has a corrected Canhydro_xylan equation. 

Version 07-08-2011: 

• Page 9: Section 11.2 includes notes on nitrogenous material in AIR. 

• Page 9-10: Section 11.3 includes pathlength in the ASL equation, which has always been 

assumed to be 1 cm, but may differ. 

• Page 11-12: Section 11.10 includes explanations and equations to correct xylan for 

branched hemicelluloses components. 

• Page 12: Section 11.13 corrects the acetic acid to acetate conversion factor.

Previous version (July 2010).